JPS63148988A - Enzymatic peracid bleaching system - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates generally to peracid bleaching, and more particularly to a novel enzymatic additive water splitting system and its use in aqueous solutions to enhance bleaching. Regarding how to.

BACKGROUND OF THE INVENTION A variety of bleaching agents have been used for numerous cleaning applications, such as cleaning and pre-cleaning of textiles, or for other applications, such as cleaning hard surfaces. In these applications, bleach is used to clean textiles,
Oxidizes various stains or stains on fabrics and hard surfaces.

Peroxygen bleaching compounds such as hydrogen peroxide, sodium percarbonate and sodium perborate have been found useful in dry bleaching compositions due to their oxidizing power.

Adding certain organic compounds, such as activators such as tetracetylethylenediamine, to perborate bleaches can improve bleach performance by forming peracids in situ.

Cleaning compositions for textiles, fabrics, and other materials with hard surfaces have been developed that utilize various enzymes to remove certain types of stains or stains. For example, protease enzymes have been found to be useful in hydrolyzing protein-based contaminants, particularly in textile cleaning.

Amylase enzymes have been found to be useful against contamination with carbohydrate substrates originating from foodstuffs, for example. Lipase enzymes have also been found useful in hydrolyzing fat-based contamination in prewash or PR soak formats.

Regarding the use of enzymes in cleaning or cleaning compositions,
Novo Industries (Nov.

IndusLrV A/S) European Patent Application Publication No. 0.
No. 130,064 relates to improvements in enzymatic additives for use with detergents in cleaning applications. The above specification described the use of lipase enzymes to achieve substantially improved lipolytic cleaning efficiency over a wide range of cleaning temperatures, including relatively low temperatures below 60°C. This document further disclosed the use of enzymes such as lipases that interact directly with contaminants or soils as a means of at least partially dissolving or mitigating contamination of such fatty substrates.

Wcyn was published on August 10, 1976.
) discloses bleaching compositions and methods of use in which an alkyl ester is said to combine with an esterase or lipase enzyme in an aqueous medium to release an acyl residue from the ester. has been done. The Weyn patent also discloses that a peracid is formed in situ when this formulation and a pre-formulation are used together.

[Problem to be Solved by the Invention] Therefore, there is a need for an improved bleach or activated oxidation system that can improve performance in low temperature cleaning conditions while retaining performance at high temperatures. It turned out that it remained as it was.

[Means for solving the problems] The present invention provides hydrogen peroxide source (J:
provides an activated oxidation system for accomplishing enzymatic perhydrolysis of substrates during production. The novel enzymes of the present invention act catalytically to promote the reaction of substrates to form peracids in situ. This new enzyme possesses excellent perhydrolysis properties and good reactivity toward triglyceride substrates even in the presence of anionic surfactants. Therefore, the enzymatic perhydrolysis system of the present invention is compatible with commercially available detergents that use anionic surfactants.

This novel enzyme is derived from Pseudomonas putida (Pseud
omonas puLIda) ATCC53552, and has the amino acid sequence shown below.

l
T.

a1mprol@wproaspthrpro11Ya
hapreph@pr.

alaw1ahaasssheasparcserga
yoretyrthrtbr ssr ser
gIn ssr glm gly p
ro ser eyes arq Itst
yrarlpre arlasa leg
glyqln*Iy 1lyval ar
q11y pra ser thr ty
r aha gIy 1eu law
ser his t4 question a1 aserhisllyphevalvalahaa
haahaI1mthrG S@rasnala@+VthrglyaN9111s
at1ewalacys0G l@wasptyrl@wvalarggluasaa
s@thrprotyr11y the sur
gly hli sat gl++
gty 11ysty sty ser4G 11. tala gIy gIn a
sp tar anl val arq
thr thrahape@IIsIlnprot
yrthrleeglylauglyhas1關asp llr alm s@r gIn
AN acc gln lln gly
pre 5et1fu'tea phm few mt ser gIy
gly gly asD thr IIs
arm phepn tyr 1mm
asn ala gin pro va+
tyr arg arq alaass
val pre wrl pha trp
wly *+++ arm ar-q
tyr va+s@r bls phe @
I@pre veasy1 11y ser gly
gly ala Iyracc lty
pn s@r thr aha trp
phe arc pha gln + shaft 21G2 O wt ase aslIII Al11 &+
l AQ Na tl+r ph tube VP
91YAl1glacyssurI11+cν5th
rsa ``hmlautrpjarvar1glyartsua
The substrate of the r@toyTell activated oxidation system is selected for an enzyme-catalyzed reaction in the presence of a source of hydrogen peroxide to form a peracid. Various triglycerides are particularly suitable for forming the substrate. Particularly preferred substrates are trioctanoin and tridecatuin.

The oxidation system of the present invention includes a source of hydrogen peroxide, which, when activated by the enzyme, binds to the substrate to generate an organic peracid in situ. For US laundering conditions, a particularly preferred organic peracid generated in this manner is beroctanoic acid.

The enzymatic perhydrolysis system of the present invention offers a number of advantages, such as the use of relatively inexpensive substrates with small amounts of enzyme to generate the product peracid. Preferred triglyceride substrates provide the ability to generate higher concentrations of peracid than provided by equivalent concentrations of simple ester substrates. The enzymatic perhydrolysis system of the present invention has been found to be highly effective in producing peracids at low temperature wash conditions.

The enzymatic hydrolysis system of the present invention consists essentially of a novel enzyme, a substrate and a hydrogen peroxide source as defined below. The invention is therefore based on peracid or perhydrolysis chemistry. Although a detailed explanation of the basic peracid and perhydrolysis chemistry is not believed to be necessary for one skilled in the art to understand the present invention, reference is made herein to the above-mentioned Shcldon N. , Lcvls), which is solely for the purpose of assisting in understanding the present invention.

In addition to the essential components of an enzymatic perhydrolysis system, including a novel enzyme, a substrate, and a source of hydrogen peroxide, the perhydrolysis system of the present invention comprises:
Preferably one selected to maintain the substrate in suspension or to solubilize the substrate in the case of an aqueous solution and to promote substrate-enzyme interaction in the presence of hydrogen peroxide from a hydrogen peroxide source. Also includes more than one type of emulsifier. It is believed that the use of this type of emulsifier of 1 fI or higher can specifically help form a liquid phase interface where the enzyme can better interact with the glyceride substrate. The perhydrolysis system may preferably include various buffers, stabilizers and other additives as described in more detail below.

In order to properly understand and interpret the present invention, including the Summary of the Invention and the Preferred Embodiments and Claims, several definitions are set forth below to clarify the use of terms used herein. Defined terms include:

"Perhydrolysis" is defined as the reaction of a selected substrate with a peroxide to form peracid and water.

In preferred perhydrolysis reactions that produce peracids, both the peroxide starting material and the peracid product are oxidants. Traditionally, inorganic peroxides have been used as oxidants, for example in dry laundry bleaches. The oxidizing ability of the peracid product makes it an effective teta stain remover in laundry bleach;
As the oxidant, the peracid product is sufficiently mild and reacts very little with the dyes and other additives used in the laundry bleach product, so that the peracid product is usually used in the laundry bleach according to the invention. It is a desirable oxidant for use as an agent. However, it is also within the scope of the invention that enzymatic perhydrolysis systems are combined with chemical perhydrolysis systems.

"Chemical perhydrolysis" generally encompasses perhydrolysis reactions in which an activator or peracid precursor is combined with a source of hydrogen peroxide. One type of peracid precursor for chemical perhydrolysis is
19 entitled "Diperoxyacid Precursor and Method"
No. 915,133, filed Oct. 3, 1986. This application describes sulfonated phenyl esters of dicarboxylic acids which are water-soluble and yield peroxyacids when dissolved in water with a peroxide source by in situ chemical perhydrolysis.

"Enzymatic perhydrolysis" is defined as a perhydrolysis reaction assisted or catalyzed by enzymes generally classified as hydrolases, and more specifically by enzymes such as those described below.

The characteristics and preferred examples of the three essential components of the enzymatic hydrolysis system are first presented below, followed by a brief description of other additives used with the perhydrolysis system, followed by a description of the enzymatic perhydrolysis system of the present invention. A number of examples are provided to illustrate decomposition systems.

Substrates As mentioned above, the substrates for enzymatic perhydrolysis systems are selected accordingly for enzyme-catalyzed reactions that form peracids in the presence of a source of hydrogen peroxide. As explained in more detail below, certain substrates typically exist as solids, in particular substrates,
Used in dry compositions containing enzymes and peroxide sources.

For such products, it is important that the dry composition exhibits a long shelf life and that enzyme-catalyzed reactions do not occur until the composition is added to an aqueous solution.

For use in laundry detergent compositions, the substrate may have surfactant characteristics, for example, such that in situ formation of peracid occurs at or near the surface of the fabric being washed. . This allows the effect of the oxidant, which is important for bleaching action, to be more effective.

The substrates for the enzymatic perhydrolysis system are various esters (RCOOR') because the new enzyme has esterase activity.
), and since the novel enzyme also has lipase activity, it may be a lipid, or both (e.g.
It may also be a combination of fatty acid ester lipid). According to the invention, it has been found that various fatty acids or glyceride type substances are particularly suitable for forming the substrate of the enzymatic perhydrolysis system of the invention.

The substances of the invention preferably have the structure I R-C-0-CH2X, where R is a substituent having at least one carbon atom, more preferably R is a phenol group, a halo group, an atom, etc. a straight or branched alkyl optionally substituted with one or more functional groups or multiple atoms, where X is a functional residue). This substrate is capable of enzymatic hydrolysis as described above, and preferably is usually not capable of substantial chemical perhydrolysis. More preferably, the functional residue consists of a functionalized polyol or polyether. More broadly, a functional residue has at least one carbon atom and at least one functional group.

Preferably, the substrate of the invention comprises essentially (i)
+M-built R2C-QC-R.

(However, R1 is C1 to C1j, and R2 is C3 to C1
□ or H, and R5 is C1 to CI2 or H), (ii) Structure R, -C-0 (CH2CH20), H (where n is 1 to 10, R, is as defined above), and (ii) structure R+CO (CR2CHO). H is selected from the group consisting of propylene glycol derivatives or propoquinylated esters, where H1 (wherein R1 and n are as defined above).

In the above preferred tgW, Ro is more preferably Cb-, C,, most preferably C7-C9, R2 is still more preferably 06-CIO or H, most preferably C7-C9 or H and R3 is more preferably C0-CIO or H, most preferably 07-C5 or H. In structure (i) above, R,, R2 and R3 may have different chain lengths and mixtures of these different glycerides are suitable for the present invention.

Glycerides are hydrolyzed when boiled with acids or bases or by the action of lipases. When using glycerides (i.e., acylglycerols), specifically diglycerides (diacylglycerols) and triglycerides (triazylglycerols), each triglyceride molecule can produce up to three fatty acid or peracid molecules in equal total number. Therefore, it is particularly preferred in the enzymatic perhydrolysis system of the present invention. The use of such glycerides is particularly effective in achieving maximum oxidizing power in the presence of a peroxide source and enzymes, as explained in more detail below.

Broadly speaking, glyceride substrates are characterized by fatty acid chains having from about 1 to about 18 carbon atoms. Low molecular weight glycerides derived from products such as acetic acid exist naturally as liquids. Therefore, incorporation of such substrates into dry compositions such as laundry detergents may require additional processing steps. However, lower molecular weight glyceride products may be more effective for high temperature laundering.

High molecular weight glyceride substrates such as stearic acid, which is characterized by a chain having 17 carbon atoms, are usually present as solids and are therefore easy to incorporate into, for example, dry laundry compositions. However, such high molecular weight fatty acid chains may not produce maximum oxidizing power according to the invention.

The most preferred form of substrate for use in the enzymatic perhydrolysis system of the present invention is trioctanoin or tridecatuine, which are characterized by fatty acid chains having 8 and 10 carbon atoms (including the carbonyl carbon), respectively. I discovered something.

These two triglycerides also tend to exist as solids and are therefore incorporated into dry compositions such as those described above. At the same time, trioctanoin and tridecatuin exhibit surfactant characteristics in aqueous solution. , readily forms peracids in situ as described above.

All of the above-mentioned substrates, including triglycerides, such as the most preferred trioctanoin and tridecatuin, are relatively inexpensive and are also important in reducing the initial bimodality of the enzymatic perhydrolysis system of the present invention. As explained below, the substrate and hydrogen peroxide source must only be present in extremely small amounts beyond the stoichiometric amount for the enzyme to generate the expected in situ peracid in aqueous solution. These are the two main components of the enzymatic water-splitting system that are required. Thus, an enzyme acts catalytically in that it is oxidized in a reaction but not consumed, but regenerated and used in further reactions.

Peroxide Source Substantially any peroxide source may be used as the oxidant thickness for the enzymatic water-splitting system of the present invention. For example, the peroxide source may consist of a perborate or percarbonate salt, such as sodium perborate or sodium percarbonate. Additionally, the peroxide source may consist of or include a hydrogen peroxide adduct, such as urea hydrogen peroxide.

Preferred peroxide sources include sodium perborate hydrate, sodium perborate tetrahydrate, sodium carbonate peroxyhydrate, pyrophosphate peroxyhydrate, urea peroxyhydroxide, sodium peroxide, and the like. A mixture of Sodium perborate hydrate and sodium perborate tetrahydrate,
Particularly preferred as an alkaline peroxide source.

The peroxide source (i.e., a compound that produces hydrogen peroxide in an aqueous solution) constitutes a peroxygen bleaching compound by itself. However, enzymatic water-splitting systems improve bleaching performance. Therefore, no further explanation of this oxidant thickness is deemed necessary except that the particular oxidant thickness is also selected to produce hydrogen peroxide in accordance with the above description.

EnzymesEnzymes are present in an unfavorable oxidizing environment when using enzymatic hydrolysis systems due to the presence of peroxides and the desired peracids. Since peroxides or peracids inactivate the enzymes used, enzymes suitable for the present invention are capable of sufficiently passing through water decomposition over the expected concentration range of hydrogen peroxide and the desired range of peracids. must be physically active.

The novel enzyme (sometimes referred to herein as "lipase 1")
omonas pwtida) and can be separated from it. Pseudomonas is a genus of rod-strapped bacteria. Several strains, including Pseudomonas putida (P, puLida), use polyoxyethylene monooleate ([Tween 80 J
, Atlas Chemical
Cal) has been shown to have a limited ability to grow on minimal media (Haue (Il.
Ove) et al., J. Gen.

Microbiol, vol. 92, pp. 234-235 (
(1976)). A novel Pscudomonas putida from which the lipase 1 enzyme can be isolated
S put4da) strain culture was obtained from the American Type Culture Collection (12301, Parklawn Drive,
Rockville%Maryland, 208
MPEP608.52) in the permanent culture collection.
1 (P), ATCC 5355
It is registered as 2.

The microorganism of the present invention is the above-mentioned Pseudomonas putida (Ps
Cudomonas putida) strains,
Natural and artificial mutant strains of the above-mentioned microorganisms can also be used. Furthermore, it is also possible to use applicable genetic engineering techniques for the production of lipases, such as the transformation of genes corresponding to the strains of the invention into other cells; Of course, other lipases are also included in the present invention.

For production, a lipase-producing microorganism belonging to the genus Pseudomonas is cultured in a conventional medium, and the enzyme can be isolated and purified. Liquid or solid media can be used. For industrial production, immersion aerated culture is preferred. Ordinary nutrient media can be used.

The incubation temperature can be varied depending on the desired microbial growth rate and is between 25" and 35°C. The incubation time can be selected as desired and is between 15 and 50 hours. Depending on the concentration of lipase or the maximum When this happens, the culture can be stopped.

Lipase accumulates in the fermentation broth and extraction of the produced enzyme from this broth can be carried out as described below.

Cells and cell debris are first removed from the whole cell broth culture by microfiltration and centrifugation, and then the lipase is adened by ultrafiltration. Excess salt and dye are then removed by dialysis or diafiltration.

The crude enzyme solution can then be purified by conventional protein purification methods. Enzyme powder can be obtained by lyophilization and used in the compositions of the invention.

Lipase 1 has the following amino acid sequence.

ala 9ra hu era so
tI1rpro v+y ala prro
phe 9'kua1aVl+Ala&SnphfAI
DaQS#Fgayoretyrthrthr se
r +er gln +er glu
gly pro sy cys arg
lletyr aQ Pre art A
Ipleu 9'y gli 91y
9'V val yqhlsprovatliel
eutrpglya=nglythrglyalagl
yprose 『thrtyralaglyleuIev
serhlstrpala$erhlsstyIlh*
valvatalaalaalaalagluthrsera
SIIalaglythr917ar9g+++set
Ievalacysle++asplyrlevval
arqgluasnas11thrpratyr+10 91Y thr tyr %er 1'V
In Ieu asn thr 9
'r 4+'9 vatgly thr s@
r gly his sur gla
gly 91Y sly 91YserII
s mt ala glV gln as
p thr arg wal arg
thr Inraha pr @ IIs
g+++ pro tyr th
r le++ gly lau
gly hisaspsirhatake11strIIsa
Qanglagb+9+VproMtphm lNwt
ser gly gly gly asp ta
r ITe ala phepre+ tyr
lee asn alm g+todoroki prm
w1tyr arg a* alaMu
wal 9rs Vat ph@trll
sly llu 'aq ar'g t
yr Vatser Mountain+11111 11+
+ 9re v41 sly %y 9+
, 9'V aha tyrar9sty l
lff ser thr aha trp phe
arg phe gin +, 2go O mtasgaspgl++atllalaaQahat
hrphtyrl'y25G ala 91+1 (ys $sr few (71
tFr %@r +ev lsw trpser
w1gjy an aPW117 1e
++ Pseudomonas ψ putida
Mutants and variants of ATCC 53552 (puLida) ATCC 53552
al selection pressureLeeh
niques), can be obtained by ultraviolet irradiation or by the use of mutagenic compounds.

They can be carried out by genetic engineering techniques, for example by transfer of plasmid DNA into multicovy hosts or by excision of the chromosomal gene encoding lipase from the cells of lipase-producing bacteria and then cloning this gene into a suitable vector molecule. It can also be manufactured by

The present invention encompasses mutants, variants or cloned strains that retain, alter or enhance the ability to produce reverse.

As further described in Example 9, FIG. 1 is a map of the 4.3 kb EcoRI of pSNE4. The box shaded with diagonal lines represents the signal peptide codon (codon-22).
~+1) and the dotted region represents mature lipase 1 voripebutide codons +1 to +258. A putative disulfide bond is shown. The measure is base pairs (bp).

The sequenced region (1363 bp Sphr fragment) is indicated by a double arrow. ATG start codon and TA
The A stop codon is also marked.

Since the preferred means of carrying out the invention (i.e. the production of reverse 1) is by cloning, the cloning and expression of reverse 1 as described in Example 9 is described herein, and surprisingly high yields were obtained in Example 9. Obtained by the treatment method.

Riverase 1 has excellent hydrolytic activity and produces peracid from substrates in the presence of a peroxide source even in unfavorable oxidizing environments. Reverse 1 also generates peracid in the presence of anionic surfactants, which typically suppress the activity of the enzyme. Additionally, Lipase] has a higher peracid/acid ratio than commercially available enzymes such as Lipase CES.

Reverse 1 can be used as a crude preparation from fermentation of Pseudomonas butig, but it can be isolated and purified from pond protein by means known in the art such as ion exchange and gel filtration chromatography. Preferably, the lipase 1 is produced in a manner that is substantially enzymatically pure. This is mainly due to the fact that the crude fermentation broth of Pseudomonas putida has been found to contain, in addition to lipase 1, another enzyme (hereinafter referred to as "lipase 2").

Although crude preparations may be utilized in bleaching agents according to the present invention, it is preferred to utilize substantially pure Lipase 1 preparations. The two enzymes, lipase 1 and lipase 2, can be separated by means known in the art, such as chromatography. They can be distinguished by the different rates of hydrolysis of p-nitrophenylbutyrate and p-nitrophenylcaprylate. Lipase 1 is produced by cloning to express this enzyme in a host organism such as E. coli and then translating the cloned lipase 1 into an octyl sepharose chromatograph as described in more detail below. It can also be produced by performing

Lipase 2 is also novel and can hydrolyze glyceride substrates and be used in applications such as fat and ura processing as a digestive aid.

Emulsifiers As with other peracid bleach products, it is generally desirable to use emulsifiers or surfactants. The use of emulsifiers is believed to be particularly important in establishing and maintaining a phase interface that promotes interaction between enzyme and glyceride substrate. Emulsifiers or surfactants are also important in establishing and retaining enzymes and substrates in the aqueous phase.

Anionic surfactants (also commonly present in commercially available detergents) may also be used. Examples of such anionic surfactants include 06-C11+ fatty acids and resin acids, linear and branched alkylbenzene sulfonic acids, alkyl sulfates, alkyl ether sulfates, alkanesulfonic acids, olefin sulfonic acids, hydroxyalkanesulfonic acids. ,
There are ammonium, substituted ammonium (eg mono-, di- and triethanolammonium), alkali metal and alkaline earth metal salts of acyl sarconates and acyl N-methyltaurides.

Nonionic surfactants are also suitable for use in the enzymatic perhydrolysis systems of the present invention. Nonionic surfactants include Shell Chemical Company (Shell Che
Neodoll (N
There is also a linear type of xylated alcohol sold under the trademark EODOL. Other nonionic surfactants have an average length of about 6 to 16 carbon atoms and an average of about 2 ethylene oxides per mole of alcohol.
~20 moles of various linear ethoxyl alcohols, with an average length of 6 to 16 carbon atoms, and an average of 0 to 10 moles of ethylene oxide and about 1 to 10 moles of propylene oxide per mole of alcohol. Certain linear and branched primary and secondary ethoxylated, propoxylated alcohols, linear and branched alkylphenoxy (polyethoxy) alcohols having an average chain length of 8 to 16 carbon atoms. and 1.5 to 30 moles of ethylene oxide per mole of alcohol, and mixtures thereof.

Other nonionic surfactants include certain block polymers of propylene oxide and ethylene oxide;
There are propylene oxide and block polymers of ethylene oxide and propoxylated ethylene diamine, and semipolar nonionic surfactants such as amine oxides, phosphine oxides, sulfoxides and their ethoxylated derivatives.

Suitable cationic surfactants include quaternary ammonium compounds, typically in which one of the groups attached to the nitrogen atom is a 08-CI8 alkyl group and the other three groups are short chains. Some are alkyl groups that may have inert substituents such as phenol groups.

Further suitable amphoteric and zwitterionic surfactants, which may contain anionic water-solubilizing groups, cationic groups and hydrophobic organic groups, include aminocarboxylic acids and their salts, amide, nocycarboxylic Acids and their salts, alkyl betaines, alkylaminopropyl betaines, sulfobetaines, alkylimidazolinium derivatives, certain quaternary ammonium compounds and certain tertiary sulfonium compounds. An example of a highly suitable amphoteric surfactant is US Pat. No. 4.00 to Jones.
Although it is described in columns 11 to 15 of the specification of No. 5,029, please refer to the above-mentioned patent specification for details.

Examples of other emulsifiers include water-soluble or dispersible polymers such as polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), methylhydroquinpropylcellulose (MHPC), or bile and other natural emulsifiers.

Other Additives It is contemplated that a wide variety of other additives may be used in combination with the enzymatic perhydrolysis systems of the present invention, depending on the particular anticipated application. For example, enzymatic perhydrolysis systems are used in a wide variety of cleaning products or compositions, such as direct bleach products, preclean products (sometimes in liquid form), and various hard surface cleansers. or may be included.

In liquid compositions, it may be advantageous to separate the hydrogen peroxide source from either the substrate or the enzyme, preferably the broth.
U.S. Pat. No. 4,585,150 (1986)
This can be done by using a dispenser divided into a number of chambers, as described in Apr. 291J).

Suitable additives include perfumes, dyes, builders, stabilizers, buffers, and the like. Stabilizers can be included to accomplish a number of purposes. For example, stabilizers may also be used to establish and maintain the effect of an enzyme on components or intermediates of the composition that are present after the composition is added to the aqueous solution. Enzymes can be inhibited from hydrolyzing substrates due to heavy metals, whitening compounds, etc., so these effects can be inhibited, for example by using suitable stabilizers commonly known in the art. , the effect of the enzyme in the composition can be maximized.

The preferred pH level in the aqueous solution in which the enzymatic perhydrolysis system is dissolved is about 8-11;
~10. Buffers can be used in the present invention to maintain a desired alkaline pH level in aqueous solutions. Buffers include all substances well known to those in the detergent industry. In particular, buffers contemplated for use in the present invention include carbonates, phosphates, silicates,
Including, but not limited to, borates and hydroxides.

The following experimental methods, materials, and results are described for the purpose of illustrating the invention. However, other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[Example] Experimental Example 1 (A) Sowing and fermentation Seed medium was mixed with 0.64'6 nutrient broth (Dil
'co)) and 1% glucose (pH 6,5). 100 ml of this medium was sterilized in a 500 ml Fernbach flask. Flasks were inoculated in loops with an overnight culture of P. putlda ATCC 53552 grown on nutrient agar and placed on a New Planswick shaker for 12 hours at 25 Orpm and 30°C. The culture incubated for 12 hours is then transferred to a 1 liter fermenter (250 ml working volume), a 1 liter biorafit fermenter (12 liters working volume 1a) or a 100 liter biorafit fermenter with a temperature control device. ,
Appropriate amounts (1 to 1096 (volume@/capacity)) were seeded in a vessel equipped with PPM, airflow and pressure regulation equipment.

The culture solution for the fermenter is 0.6% nutrient broth (Difco),
It contained 0.396 apple cutin and 0.29o yeast extract, and the initial pH was 6.5. This culture solution was adjusted to pH 6.
, 8 and sterilized for 40 minutes before sowing. Bacterial growth and enzyme production continued in the fermenter for 12-15 hours.

(B) Enzyme recovery by microfilter The crude fermentation culture was first filtered into two Romicons (R(+1lic).
on) Amicon (
Cells were removed by filtration in an Amicon) unit. Enzyme remaining in the retentate bound to cutin was removed by centrifugation. The total recovery rate was 90%.

(C) Concentration and permeation of total cell filtrate: The filtrate collected from the Vamicon unit was concentrated on two Amicon Pm 10 module Amicon ultrafiltration units to a volume of 3 liters. Concentrated. The concentrated material is then added to 20 liters of 0.
Salts and dye were removed by dialysis against 01M phosphate buffer, pH 7.5. The average recovery rate at this stage is approximately 8.
It was 0%.

The total activity of this crude preparation was 8-. It was 68 x 106 units. The unit of lipase activity is 0.1ffif
2.0 in O, 1 M Tris-HCl buffer, pH 8.0 containing fi96 Triton (Trlton >
When incubated with mM p-nitrophenylbutyrate at 25°C, the absorbance at 415 nm was 1.
Defined as 2 of the enzyme increasing 0/min.

Example 2 Lipase activity after ultrafiltration and diafiltration reaction conditions were 0. The binding and cumulative reaction rate of three p-nitrophenyl substrates was studied for the crude preparation of Example 1(C) in 0.1 M Tris containing 1 wt% Triton X-100, pH 8, 0, 25°C. did. The substrates are p-nitrophenylcabrilate, p-nitrophenyllaurate and p-nitrophenylcabrilate.
-nitrophenyl palmitate, and the data are shown in Table 1.

Table-1 Substrate K,, V,, (μN1) (μmol/min/ff1g protein) PNPC
2+4 802 PNP! , 167 2+4
PNPP 183 112 Example 1
The preparation of Example 1(C) was used in various experiments further described below to demonstrate its usability in the enzymatic perhydrolysis system of the present invention; however, the preparation of Example 1(C) 2 “Lipase 1
” and an enzyme named “lipase 2.” Lipase 1 is a better perhydrolase, and particularly preferred embodiments of the invention employ substantially enzymatically pure lipase 1 preparations. Isolation and purification of the preparation of crude Example 1 (C) is described in Example 3, where complete separation of lipase 1 and lipase 2 is preferred to obtain substantially enzymatically pure lipase 1. Example 4
and a lipase 1 preparation (ie, a preparation for analytically pure sequences) is described in Example 5.

Example 3 Lipase 1 was first introduced into Pseudomonas putida (Pseudomonas putida).
domonas putida > DEA from fermentation broth
E-Sephacryl chromatography followed by partial purification by Sephadex G-100 gel a perchromatography. The DEAE column was equilibrated in 10 mM sodium phosphate buffer, pH 8, and the crude protein was added to the column in the same buffer.

PNB (p-nitrophenylbutyrate) hydrolase activity that did not bind to the column was related to lipase 1. Lipase 1 thus obtained from the DEAE step
was chromatographed on Sephadex G-100 in 10 mM sodium phosphate buffer, pH 8. Lipase 1 elutes from this column as a discrete peak, and P
Identified by NB hydrolase activity and perhydrolysis activity.

Example 4 Lipase 1 can be completely separated from lipase 2 by chromatography on a hydrophobic resin. The enzyme solution of Example 1(C) was adjusted to 0.5 M NaCl after ultrafiltration and diafiltration and equilibrated in 10 mM Tris(C1), pH 8, 0.5 M NaCl. In addition to Sepharose column,
Unbound protein was removed by washing. The following cleaning solution was used. 10mM Tris, pH 8 and 2-1 urea,
and 10mM sodium phosphate, pH 8, and lQ
After washing with mM phosphoric acid, pH 8 and 0.5M NaCl, the column was developed with a linear gradient to 5096 n=propertool. The column fractions were then assayed for p-nitrophenylbutyrate (PNB) and p-nitrophenylcabrilate to determine lipase activity. The two types of lipase are clearly separated, and in fraction 32, PN
The ratio of B/PNC is 4.6, and in fraction 52 PNB
/PNC ratio was 1.40. These were named lipase 1 and lipase 2, respectively.

Fractions from this column were further analyzed by SDS gel electrophoresis. By this analysis, the two lipase activities showed a molecular weight band of 30.000 with characteristics of prokaryotic lipases, and furthermore, lipase 2 migrated as a doublet and was clearly separated from the single band of lipase 1. . Prior to sequence analysis, these two partially purified enzymes were separated from high and low molecular weight contaminants by reverse phase chromatography.

Example 5 Prior to sequence analysis, the partially purified material of Example 3 was further purified by chromatography on a 4.8xb (SynChroa+Pak) C4 reverse phase HPLC column. The system was injected with 0.605% triethylamine (TEA) at 0.5 ml/min.
) and 0.05% trifluoroacetic acid (TFA) (
Equilibrated in solvent A). 1+ng of lipase 1 to 10
The diluted solution to 0 μg was injected into the column and mixed with solvent A and 0 μg.
0.0% of the compound with a gradient with n-propatool (solvent B) containing 0.05% TEA and 0.05% TFA. 5m
It eluted at 1/min (B/min).

Typical slopes are +5% for O~20%B and 60%
%B was 100.5%B/min. All lipases are inactivated in this HPLC solvent system. Approximately 3
The protein peak eluting at 5% Solvent B (Lipase 1) or the protein peak eluting at about 3996 Solvent B (Lipase 2) was collected and used for subsequent sequence analysis and preparation of CNBr fractions.

Practical Example 6 A cyanogen bromide peptide fraction for amino acid sequence analysis was prepared and purified as follows. A portion of the pooled lipase 1 of Example 5 was subjected to SpeedV
ac) Dry in a centrifuge and then redispersed in 8M urea containing 8896 formic acid at a concentration of 10Ll1g/ml.

The solution was mixed with a 200 mg/ml volume of CNBr in formic acid and incubated for 2 hours at room temperature in the dark. The product was then desalted on a 0.8 x 7 cml BF-trisacyl GFO5 (rough) column to 40% Solvent B.
:50S;6 After using solvent A (see above), reverse phase analysis was performed. The peptides were first separated by reverse phase using the same protocol described above for lipase 1 purification. However, solvent B was changed to 3596 brovanol = 65% acetonitrile (containing TEA and TFA). The initial digest and post-chromatography peaks were also analyzed on SDS/urea/pyridine gels followed by silver staining.

Two peaks were selected from the chromatogram and rechromatographed, in this case on a 0.48x25e+n Zinchrome Pack 04 column, using the same conditions as above. After rechromatography, the purified peptides were immobilized and sequenced.

Example 7 (Same as Example 4) The purified fractions of Lipase 1 and Lipase 2 from an Octyl Sepharose column were treated with Solvent A (0,05'6 triethylamine and 0
．． Chromatography was performed (as in Example 5) with 3 volumes of 0.05% trifluoroacetic acid. As described in Example 4, the purified proteins were analyzed by SDS gel electrophoresis and then pooled to compare the CNBr fraction and N-terminal amino acid sequences of lipase 1 and lipase 2, respectively.

Example 8 Specific activity of lipase 1 The specific activity of lipase 1 was determined using the enzyme purified in the same manner as in Example 4. The specific activity of substantially enzymatically pure lipase 1 is 3750, as defined in Example 1(C).
It has a tag protein at position 1.

Example 9 Pseudomonas pu
tlda) strain (ATCC53552), 200ml/L
Grow overnight at 37°C in B (Luria broth) medium.

Cells were collected by centrifugation and high molecular weight total DNA was extracted.
Birnboim et al., Nuclel
cAcids Res, vol. 7, 1513-1523
(1979). The DNA was digested to completion with EcoRr, EcoRI, T4 DNA ligase, and ligated to a preparation of plasmid pBR322 (ATCC 37017), which had been dephosphorylated with bacterial alkaline phosphatase. All enzymes used for DNA manipulation were purchased from the manufacturer (Niney England Biolapse).
ew England B111abs) or Hetesda Research Laboratories (BeLl+esd
aRe5earch1, aboraLories)
) was used according to the instructions. The bound DNA was transferred to E. coli 2Q4 (ATCC31
445) was used to transform and anbinlin-resistant colonies were selected. Therefore, approximately 2xlO' transformants were collected (5x103 per plate). Add 4-methylumbelliferyl butyrate (50 r
A solution of 10 mM in nM Tris-HCl, pH 8.0) was added and irradiated with an ultraviolet lamp (wavelength 340 nm). Colonies that hydrolyzed the substrate and released highly fluorescent 4-methylumbellifolone exhibited a strong blue color. Thirteen positive colonies were obtained using this method. From each of these positive colonies, a plasmid minipreparation (IIiniprcp) was prepared by the alkaline sys- tem method described by Birnboim (supra). Each plasmid was digested with EcoRI, and the resulting fragments were published in Mniatls et al., "Molecular Cloning, Laboratory Manual," Cold Spring Harbor Laboratory.
(Harbor I, aL+oraLory), Cold Spring, New York (1982). Most of the plasmids are single-inserted 4
, contained a 3 kb fragment. Others contained other fragments outside of this one.

This result suggested that all positive colonies arose as a result of expression of a common cloned gene contained in a 4.3 kb fragment. One of the plasmids containing only the 4.31cb fragment and designated pSNE4 was selected for further analysis.

Plasmid pSNE4 was digested with various restriction enzymes containing a 6 bp recognition sequence. These enzymes were used singly or in pairs. By analyzing the size of the fragments resulting from these experiments, the 4.3 kb EcoRI p
A preliminary restriction endonuclease cleavage map of the SNE4 insert could be purified. This map is the first
As shown in the figure.

Several subfragments of the EcoRI insert of plasmid pSNE4, at least 840 bp, were subcloned into pBR322 to determine whether they contained functional genes. The plasmid extracted containing the functional lipase gene contains psNEsl.
2, from the EcoRI insert of pSNE4.
Contains a 3kb EcoRI/SalI fragment (
(See Figure 1 for the map layout of this fragment).

The inserted fragment of psNEsl was digested with different restriction enzymes, and the resulting small fragment was purified by Roberts (RoL+erLs), Nucleic Ac1ds.
Res,, Volume 12, Addendum r167-r204 (1
Sanger et al., Proc, Natl, Acad, Sc1.
The sequence was determined by the dideoxy chain termination method of USA, Vol. 74, pp. 5463-5467 (1977). sp
The sequence of 1.36 kb of DNA between the h1 sites (see Figure 1), when translated in all possible reading frames, yields the protein as determined by the direct amino acid sequence (residues 1-16). The N H2 terminal amino acids represent a large open reading frame. This open reading frame also covers two other directly sequenced peptides (residues 94-105 and residue 1).
73 to 190). The methionine at position -22 appears to be the start codon since it begins the code for a highly hydrophobic region typical of signal peptides. This signal peptide is likely to be cleaved during the secretion step after the alanine at position -1. The open reading frame ends at position 259, indicating that the encoded mature protein has 258 residues.

In order to achieve controlled expression of the Pseudomonas putida (P, putlda) lipase gene in E. coli (E, colt), before the ATG start codon,
Adelman (Adc) in bacteriophage M13
DNA, Volume 2, 183-193
After initially introducing the XbaI site by site-directed mutagenesis of DeBoer et al. (1983), Proc, Na1l, Acad, Sci, US
A, vol. 80, p. 2125 (1983) strong ta
The vector was cloned into an expression vector containing the cll promoter. This was done by first digesting psNEsl with sphI.

2.4kb of 5phl containing the entire lipase coding sequence
The fragment was isolated and M 13 at its 5 phl site.
m p 1.9 (RF) and the mixture was transformed into E. coli (IE, coli) JMIOI (
ATCC33876) was used for transfection. Clear plaques were collected to prepare bacteriophage (template) DNA in which the Sphi fragment was present in a counterclockwise orientation. A partially complementary single-stranded fragment of DNA consisting of 50 nucleotides immediately 5' of the lipase IATG initiation codon.
A product containing Xbal at the site was synthesized. This 50-mer (before the ATG start codon)
-27 nucleotide position to -9 position and +1
Capture template DNA between positions (A of ATG) and +20. However, between positions -9 and +1,
Sequence of the original lipase promoter region 5'-AAC
CTTCG-3' was a change to 5'-TATCTAGAATT-3' of the tacll promoter. Mutagenesis was performed.

300 plaques were isolated using a 32p-labeled rigid oligonucleotide (5'-ATGAGG) spanning the area of change.
Screening was performed by hybridization with TATCTAGAATTATG-3'). A positive hybridizing clone RF was prepared and Xba
Cleaved with I and 5phl. lkb x containing gene
The baI/5phl fragment was isolated and deBoer (
deBoer) (supra).
907tacII was digested with Xbal and 5phl to generate 4.1kbXba containing the taclI promoter.
The t/5phl fragment was isolated and ligated into the vector and the ambinlin resistance gene. J Ml 01 cells were then transformed with the ligation mixture. (Plasmid psNtacll (see Figure 2)
Ambicillin-resistant colonies were selected.

To measure the levels of cloned lipase 1 synthesized by E. Coil, J.M.
IOI/pSNt ac II was grown in 20 ml of LB media supplement with 1 mM isopropyl-B-D-thiogalactoside (IPTG) at 37°C for 10 hours. 294/1) BR322 was used as a negative control. Cells were separated from the culture supernatant by centrifugation and then fractionated into Koshland.
and) the cell periphery of (above) and It! /! It was designated as II cytoplasmic component. Each fraction was tested for activity by p-nitrile phenylbutyrate hydrolysis. β-lactamase (pericellular marker) and β-galactosidase (cytoplasmic marker) should also be measured.
) et al., Proc.

Natl, Acad, Set, USA, Vol. 81, pp. 2645-2649, 1984), the effectiveness of the cell fractionation method was confirmed. Most of the lipase 1 activity (74%) was present in the culture supernatant. The majority of cell-associated enzymes were found in the cell wash fraction (17% of the total amount), with smaller amounts present in the pericellular (2%) and cytoplasmic/membrane (7%) fractions. In the 294/pBR322 negative control culture, no lipase 1 activity was present in any fraction.

E. coli harboring the plasmid psNtacll (
The broth resulting from the fermentation of strain JMIOI (E, coll) was adjusted to 0.5 M NaCl, eliminating the propatool gradient, lOm~1 sodium phosphate, pH 8.0,
Pseudomonas putida (P, pu
tida) was purified by Octyl Sepharose substantially as described (Example 4). The isolated product (cloned from the gene expressing the enzyme) was analyzed by SDS gel and migrated similarly to the lipase 1 product isolated from the original Pseudomonas puLida strain.

Example 10 A cyanogen bromide fragment from cloned lipase 1 was prepared as follows. The product obtained by Octyl Sephas purification of the cloned product (Example 9) was diluted with 3 volumes of solvent A and subjected to short C4HPL.
On the C column, Pseudomonas putida
Lipase 1 and Lipase 2 isolated from P. onas puLida were purified as described.

The products were analyzed on an SDS gel.

Example 11 CNBr fragment of lipase 1 in Pseudomonas putida (P, puLida) and cloned lipase 1 in E. coli
compared the CNB r fragments from. HPLC-purified lipases 1 and 2 from Pseudomonas and cloned lipase 1 were each hydrolyzed by CNBr as described in Example 6 above. The products were analyzed by SDS/urochord/pyridine electrophoresis. This result clearly shows that the cloned protein is lipase 1. (Examples 4-5
) Pseudomonas putida (P, putida)
) Lipase 1 isolated from a living organism is isolated by the same chromatographic method (as in Example 4); (b) the N-terminus of Lipase 1 isolated from any living organism is The amino acid sequence of (c) is also the same.
The CNBr fragment pattern clearly identifies lipase 1 and lipase 2, and Pseudomonas putida (P
, putlda) or E. coli (E, co.
li) the CNBr fragments of both lipase 1 are identical and (d) the p-
The activity ratios of nitrophenylbutyrate and p-nitrophenylcabrilate MW are the same and (e) the hydrolysis/perhydrolysis ratio with tricabrilin as substrate is the same for lipase 1 isolated from both organisms. By the criterion of being the same as the ratio, E. Corey (E, c
Cloned lipase 1 isolated from oli)
was shown to be the same.

Example 12 Method for Measuring Peracid Generation A method for measuring peracid generation was developed by observing the oxidation of 0-phenylenediamine (rOPDJ). Oxidation of OPD by peracid.

Much faster than with H20□, 458nm
Increases absorption in. The assay consists of withdrawing 0.1 ml of the reaction mixture to be tested, adding 0.2 ml of OPD solution (in some cases OPD is added to the initial reaction mixture), incubating for 5 minutes at room temperature, and adding 0.9 ml of the reaction mixture to be tested.
of CHCl3/CH,OH (1: 1 (volume/volume vi
)), centrifuged for 1 minute, and read the absorbance at 458 nm. The standard plot (for Cs peracid) was linear at least between 36 ppm peracid.

Example 13 shows that a "crude" preparation containing a preferred enzyme ("Lipase 1") and another less preferred enzyme ("Lipase 2") was prepared in the presence of 30 ppm peroctanoic acid or 800 ppm or less hydrogen peroxide. Has acceptable hydrolytic activity.

Example 13 Stability of lipase in the presence of hydrogen peroxide and peracids (as in Example 1 (C)) Lipase 1 and Lipase 2
a predetermined amount of the enzyme preparation of Example 1 (including a mixture of
mg/ml, 0.5%; trioctanoin, 10
pH 10 for each of the five samples in combination with 0mM NaCl and 10mM sodium phosphate.
A 2 ml reaction volume was formed with . The reaction mixture for each sample was kept at 30°C. Five samples were tested for hydrolytic activity as described below. 30 ppm of bero-octanoic acid was added to one sample, and the hydrolytic activity of the enzyme was determined in the same way as the hydrolytic activity of the control (no bero-octanoic acid was added) (N a
micromoles of OH 7 min). Hydrogen peroxide was added to the two others (400 ppm and 800 ppm, respectively) and the hydrolytic activity was measured in the same way as the hydrolytic activity of the control. The data are shown in Table-2.

Table-2 Hydrolysis active sample Additive (1 mole of NIOH
．． (control) Oppm Beroctanoic acid 0.482 30pp
m Beroctanoic acid 0.2423 (control) Oppm Beroctanoic acid 0.514 400p
pmH2020,4135500I)pmH2(h
Although the hydrolytic activity of this crude enzyme preparation was reduced by the presence of bero-octanoic acid and hydrogen peroxide, as shown by Table 2 of Table 2, this enzyme preparation remained stable even in an unfavorable oxidizing environment. It appeared to be fully hydrolytically active.

Similarly, the same amount of commercially available enzyme (K-30) was added to 0.5f
2 ml each containing fl volume % trioctanoin, 100 mM NaCl and 10 mM sodium phosphate
The reaction volume was tested by adding 400 ppm or 800 ppm hydrogen peroxide to the reaction volume. The hydrolytic activity was measured in the same way as the control hydrolytic activity (N
micromoles of aOH 7 min). Table-3 shows data for comparison with Table-2.

Table-3 Hydrolysis active sample additives
(HOH molar fraction/minute) 6 (Control) OppmH2020,223 7400pI) mH2020,135 8800ppmH2020,05G Comparing the data in Tables 2 and 3, the enzyme of the present invention has a higher oxidation rate than the conventionally known enzymes. It is found to be quite stable (low sensitivity) in the sexual environment.

Example 14A shows that a pure preparation of the preferred enzyme (lipase 1) was not inactivated and the 4-
It shows that it has excellent hydrolytic activity in the presence of 7 ppm of bero-octanoic acid. Example 14B shows the effect of pH on the perhydrolysis/hydrolysis ratio.

Example 14A Stability of Lipase 1 Another enzyme preparation similar to Example 4 (substantially pure Lipase 1) was tested both in the presence and absence of hydrogen peroxide. The hydrolysis rate is micromoles/
Measured as ml/10 minutes.

Each sample had a substrate of 1.0 wt.% trioctanoin and 4Il+g/ml of 0-phenylenediamine (OP D) emulsified with 0.1 wt.% sodium dodecyl sulfate (rSDSJ'). The reaction volume for each sample was 2 ml and the temperature was 27°C. Substantially pure Lipase 1 was not inactivated by the presence of 400 ppm hydrogen peroxide or the presence of peracid generated by perhydrolysis.

Example 14B Effect of pH An enzyme preparation similar to Example 4 was tested under certain reaction conditions as described below. 1 wt% trioctanoin in 0.1 wt% SDS, 1 μg/ml enzyme, 1 g/m at 21°C
1 OPD, reaction volume at 27°C is 5 ml. The pH of each reaction volume was adjusted to a value between 8 and 11, and the perhydrolysis and hydrolysis values were measured as micromoles/ml/5 min. The optimum pH appears to be a pH of about 10.

Example 15 shows that the new enzyme has greater enzymatic reactivity towards lipids than towards esters.

Example 15 Generation of Peracid on Various Substrates Each sample contained 380 ppm H202, 2o+g/ml OPD in 40 mM sodium phosphate buffer.
, 1 μg/ml enzyme (prepared as in Example 1, including controls for Lipase 1 and Lipase 2), 1 m;%
(as an emulsion in SDS with a ratio of substrate <I & quality/SDS of 10=1). pH is 9. 0 (p
Hstat), the temperature was 27° C., and the reaction volume was 5 ml. Table = 4 micromoles/ml/10
Data shown in minutes.

Table 4 Hydrolysis Perhydrolysis Substrate (Production of acid) (Production of peracid) Trioctanoin 4.24 0.42 Methyl octanoate 0.88 0.11 As is clear from the data, the new enzyme ( higher reactivity towards triglyceride substrates (compared to moderate esters);
It has been shown to be a lipase.

Many commercially available enzymes are inhibited by the presence of anionic surfactants. In fact, anionic surfactants such as SDS are usually used in processing methods such as SDS electrophoresis.
It is used to solubilize proteins by binding to protein molecules, converting them into rods, and masking their original charge with the negative charge of SDS. Since many, if not all, commercially available detergents contain anionic surfactants, the ability of enzymes to maintain the activity of detergents over a period of time is an important advantage. .

Example 16 shows that hydrolytic activity is retained by enzymes of the invention in the presence of anionic surfactants, whereas activity is inhibited by comparison commercial enzymes.

Example 16 Sonoji composition and/or except that the enzyme is specific
Alternatively, samples with reaction conditions were prepared.

The comparative sample was Asbergullus niger (Aspcr-
gillus nlger) and Amano (Am
-30 was prepared using 8.7 μg/ml of commercially available lipase from Ano. The enzyme preparation of the present invention was similar to Example 1 and contained 16.8 μg/m
1 (approximating the hydrolysis rate of -30 for lipase). Each sample had trioctanoin as an emulsion with SDS at an fLl ratio of 10:1. Sodium phosphate buffer (10mM) is present and pH
was 10, the temperature was 25° C., and the reaction volume for each sample was 2 ml. Data showing the hydrolysis of trioctanoin for each enzyme in the presence of SDS is shown in Table-5.

Table-5 Substrate hydrolysis Substrate j! (wt%) (jl of 0,1NN10H
l/min) 0 for the enzyme of the present invention, old 2 0.05 7 0.1 +1 0.5 H t, o 12 Substrate hydrolysis Amount of substrate (wt%) (all/min of 0,1N hOH
) In case of commercially available enzymes 0.01 1 0.05 7 0.1 7.5 0.53 1.0 1.5 As is clear from the data in Table 5, two different enzymes have 0.05 A substrate weighing 96% is hydrolyzed to a nearly fixed degree, but the amount of substrate is approximately 0.0%. The commercially available enzyme was inhibited by increasing amounts of SDS when increasing above 1% by weight trioctanoin.

That is, the perhydrolysis rate was relatively low at -30 for lipase. In contrast, the enzymes of the invention were substantially unaffected by the presence of anionic surfactants.

Example 16 except for the emulsifier (polyvinyl alcohol)
In a similar test, both the commercially available lipase-30 and the enzyme of the invention showed good hydrolysis rates.

Example 17 Generation of Peracids by Enzymes for Comparison Similar to Example 3, generation of peracids by M
The peracid generation of two commercially available enzymes in the presence of 0.5 wt% SDS was compared.

Each test sample contained 480 ppm hydrogen peroxide,
6μg/ml enzyme, 5% by weight and 0.5 balls amount?・6
of trioctanoin substrate in SDS.

The constant pH value was 10 and the temperature was 25°C.

Table 6 shows the state of perhydrolysis of the enzyme of the present invention.

Table 6 Time (min) Peracid generated (pprA) 23.9 47.2 89.0 10 9.9 In contrast, Pseudomonas fluorescens (Pseu
domonas I'1. The amount of peracid generated using the commercially available lipase CES (said to be derived from ) and marketed by Amano remained virtually constant and was low (approximately 0.5 ppm peracid) compared to the commercially available The amount of peracid with Lipase K was virtually constant and even lower (ca. 0.
3ppm peracid).

The ratio of perhydrolysis to hydrolysis is extremely important. It is desirable to have as high a conversion rate of substrate to peracid as possible. The enzyme of the invention has a higher peracid/acid ratio than commercially available enzymes such as lipase CES. This means that the enzymes of the present invention utilize substrate more efficiently for the generation of peracid and therefore require less substrate to be present in the bleaching composition.

Example 18 400 ppm hydrogen peroxide, 0.12 M HPO4-'
Perhydrolysis and hydrolysis were measured in samples containing a commercially available lipase (CES manufactured by Amano) or an enzyme of the present invention (preparation of Example 1 (C)) at various concentrations. . Perhydrolysis was determined by thiosulfate titration with a 14 minute reaction. Hydrolysis was measured by continuous titration using a pH stat at the same time. The second substrate was P with 12.5% Wffi% trioctanoin.
VA was 0.7'lj1%. The enzyme of the invention hydrolyzed less substrate and delivered more peracid, indicating more efficient substrate utilization than that of lipase CES.

The crude preparation shown by Example 1(C) was found to synthesize two enzymes named "Lipase 1" and "Lipase 2". For the trioctanoin substrate, one enzyme has a different perhydrolysis/hydrolysis ratio than the other, with lipase 1 being better at perhydrolysis compared to lipase 2. Example 19 shows these ratios.

Example 19 Four samples were prepared, each containing 1% by weight of substrate, 0 and 1% by weight of surfactant (SDS or PVA).
, H202, 400 ppm and OPD 4 mg/ml. The reaction volume was 2 ml, the pH was 9.0, and the temperature was 27°C. Lipase 1 (preparation of Example 3 or preparation of Example 9) or Lipase 2 (Example 4) was added to the samples. The data are shown in Table-7.

Table 7 Lipase 1 Lipase 2 (p-nitrophenyl (p-nitrophenyl hyperhydrohydration H/surfactant butyrate subtraction water decomposition unit)
Butyrate hydrolysis level) Hydrolysis ratio SDS
4 0
0.19PVA 4
0
0.14SDS 0
20 0.010P
VA 0 20
0.011St)S
L2 0
0.12PVA 12
0
0.076 As is clear from the data in Table 7, lipase 1 is more sensitive to trioctanoin substrate than lipase 2 in the presence of anionic surfactants.
,, is an extremely good perhydrolase.

Example 20 shows that using excess of the novel enzyme increases hydrolysis but not peracid. Also, °
This example demonstrates effective utilization of the substrate.

Example 20 Effect of Enzyme Concentration on Perhydrolysis/Hydrolysis Ratio The substrate was 1 wt% trioctanoin emulsified with 0.1 wt% SDS and 4 mg/ml of 400 ppm H202.
ml of OPD, pH is 9.0, temperature is 25.
The reaction volume was 5 ml. Using three different dishes of enzyme (preparation of Example 4), the results are shown in Table-8 Enzyme Level Hydrolysis (micromoles/ml) (
PN13”, unit/ml) 5 minutes 10
Minute 3 1.7 3.2G
3.5 5.912
G, l 10.1 Overforce water splitting (mic 0 mol/m1) 5 minutes 10 minutes3 0.42 0.546
0.51 0.7g120.
57 0.87 The ratio of perhydrolysis to hydrolysis after 5 minutes of 1p-nitrophenylbutyrate hydrolysis is 0.25.
0.15 and 0.09, and after 10 minutes they were 0.17, 0.13 and 0.09, respectively. Therefore, lower amounts of enzyme were more efficient.

Lipase 1 and lipase 2, when separated, were found to have quite different hydrolysis rates for p-nitrophenylbutyrate and p-ditrophenylcaprylate. These two novel enzymes can therefore be distinguished by the ratio of hydrolysis of p-nitrophenylbutyrate to p-nitrophenylcabrilate, as in Example 21.

Example 21 Reactions were carried out in 0.1M Tris-HCI, pH 8.0 and 0.1M Tris-HCI, pH 8.0.
1% by weight Trlton
II & Ilaas) at 25°C.

The hydrolysis rate of 2.0 mM p-nitrophenylbutyrate (PNB) for lipase 1 (preparation of Example 3) was 0.60 (H〇D415 nm/min), and 2.0 mM p-nitrophenylbutyrate (PNB) The hydrolysis rate of phenylcabrilate (PNC) was 0.09 and the PNB/PNC ratio was 7. In contrast, the hydrolysis rate of PNB for lipase 2 at the same concentration is 0.54, and the hydrolysis rate of PNC at the same f-rate is 0.44, with a PNB/PNC ratio of 1. Ta.

Novel enzymes, such as the presence of anionic surfactants,
Commercially available enzymes have been shown to produce peracids in the presence of a wide range of surfactants under unfavorable conditions.

Example 22 Perhydrolysis activity of lipase 1 and two commercially available lipases A sample containing either the novel enzyme (preparation of Example 1(C)), commercially available lipase K or commercially available lipase CES was treated with a substrate (triopase). (cutanoin), hydrogen peroxide and a control of surfactants (anionic and nonionic) dissolved in an aqueous solution. The solution was at room temperature and the pH was 10.0. As shown in Table-9, perhydrolysis was calculated as ppm number after 14 minutes.

The enzymes of the invention have been shown to provide very good perhydrolysis of anionic surfactants in the presence of detergents.

The above examples are based on the use of triglyceride substrates with functional group (i) as the preferred substrates mentioned above. Other glycerides included in the same functional group,
The triglycerides in the above examples can be substituted. At the same time, substituting the triglyceride substrate in the above examples with additional substrates as described above, in particular substrates encompassed by the preferred functional groups of ethoxylated esters (ii) and propoxylated esters (iii). You can also do that.

Furthermore, the above-mentioned preferred functional substrate groups (i), (ii)
With respect to and (ii), examples of specific substrates in the first functional group are clear from the examples above.

Examples of substrates from the other two types of groups are illustrated by the propoxylated ester synthesis method described in Example 23.

Example 23 A method for preparing propylene glycol monoester of carboxylic acid has the following steps.

(1) Salt Formation and Desalting One equivalent of carboxylic acid and 0.0 g equivalent of sodium carbonate were mixed in a round bottom flask equipped with a magnetic stirrer bar and a heating oil bath. The slurry was heated to 150° C. for 1 hour under vacuum with constant stirring to effect dehydration. The vacuum was released and the reaction was cooled to room temperature.

(2) Mix the cooled acid/acid acid solution from esterification step (1) with about 6 dishes of propylene oxide and heat at about 60° C. under reflux on a mild oil bath for about 8 hours. Then, an esterification reaction was performed. (Completion of the ester reaction was confirmed by a gas chromatography monitor.) (3) The reflux condensate was removed and excess propylene oxide was distilled off. Approximately 200 ml of diethyl ether per 100 mmol of acid is added and the resulting solution is diluted with 2 ml of diethyl ether in a branching funnel.
The mixture was extracted with 5% sodium carbonate. Next, 1 volume of saline solution was added. Dry the ether layer over sodium sulfate and
When filtered and evaporated in a rotary evaporator,
An ester product (typically about 90% purity) was produced.

Other examples of functionalized substrates with functionalized substrate groups (i i) and (i i i) can be produced by similar processing methods.

Example 24 describes a method for decontaminating some preferred compositions.

Example 24 Diagnostic fP values of oxidant performance were performed on 100% cotton swatches stained with crystal violet as described below.

Crystal violet (0,125g) in 1 part distilled water
．． Added to 25 liters. Add 100 2" x 2" undyed 100% cotton swatches to the solution and
Stir for hours. The cotton swatches (dyed with crystal violet) were removed from the dyeing solution and washed repeatedly with cold tap water until the effluent was almost clear.

Place each dyed swatch on aluminum foil and
Wipe with paper towel and air dry.

Three preferred compositions of the present invention are: AaJL, similarly corresponding control compositions were prepared. Three inventive compositions and three corresponding control compositions were
Each was used to wash dyed cotton swatches, and the stain removal performance of each was determined in Table 10. Composition of the present invention (a) Stain removal rate (%) 0.06% by weight Trioctanoin 63.60.04
Heavy weight sodium dodecyl sulfate 100p100pp 02 1μg/ml lipase 1 20μM EDTA (pH=10.5) Control composition (a) 0, 06ffl volume % trioctanoin 50.60
．． 04 weight% sodium dodecyl sulfate 100 p pmH202 20 μM EDTA (pH-10,5) Composition of the present invention (b) 0.06% by weight trioctanoin 80.40.04
Sodium dodecyl sulfate by weight 200 ppmH, o2 1 meg/ml lipase 1 20 tzMEDTA (pH-10,5) Control composition (b) 0.06 weight by weight 96 trioctanoin 69.80.0
4% by weight Sodium dodecyl sulfate 200 ppm H202 20 g MEDTA (pH-10,5) Composition of the invention (c) 0.02 weight 96 trioctanoin 67.40.0
Double m96 sodium dodecyl sulfate 50pprnH202 5μg/ml lipase 1 2 Q 11~IEDTA (pH-10,5) Control composition (c) 0.02 mass% trioctanoin 52.20.02
Weight % sodium dodecyl sulfate 50'ppinH20z 20μM EDTA (pH-10,5) As is clear from the data in Table 10, the composition of the present invention has no control effect even if the control composition contains a peroxide component. It provided better stain removal ability compared to the composition. These improved T9 stain removal abilities are due to the enzymatic perhydrolysis system, and it is quite surprising that such performance occurs in the presence of anionic surfactants that inhibit many well-known commercially available enzymes. It is.

The flow-through water splitting or activated oxidation systems described in various of the above examples can be used in combination with any of a variety of detergent compositions typically used in textile selection. For example, typical heavy-duty powder detergents used in US wash layers include anionic and/or nonionic surfactants, phosphate or non-phosphate builders, buffers, and brighteners, fragrances, , including related additives such as proteases, etc. The floating water splitting system of the present invention can be used by itself or as a small portion of such typical powdered detergents.

Typical heavy-duty powder detergents used in Europe have approximately the same nominal composition as those used in the United States, but the working concentration of the product is typically In washing machines it is usually a 1.2% solution. A preferred composition of the marine water-splitting system of the present invention when formulated with a typical detergent composition is about 3 to 30% peroxygen, about 0.6% to about 12% by weight. 6 substrate and about 0.001 to about 0.7 weight? Mono lipase 1.

For use in a typical U.S. wash layer (where the detergent typically does not contain oxidizing bleach and decontaminating enzymes), in addition to the detergent, an oxidant (typically sodium perborate or It is common practice to use products containing sodium percarbonate) and enzymes (proteolytic and amylolytic enzymes). These oxidant-containing products are typically washed in a washing machine at a product working concentration of about 0.17% solution.
It is formulated to generate about 25-50 ppm of atomic oxygen (hydrogen peroxide). The ship water decomposition system of the present invention,
When used with detergents in wash water at temperatures of about 70 to about 100 centimeters, the preferred composition of the floating water splitting system of the present invention preferably comprises a hydrogen peroxide source, a substrate, and a lipase 1
in a weight ratio of about 2400:200:1 to 48:20:1.

A particularly preferred enzymatic hydrolysis system of the invention comprises sodium perborate tetrahydrate, trioctanoin and lipase 1-a in an ff1lit ratio of 95:39:1. Such laundry additive compositions contain about 25 to 50 ppm atomic oxygen (hydrogen peroxide), 2 to 20 ppm atomic oxygen (theoretical peracid), and 0.5 to 50 ppm atomic oxygen (theoretical peracid). lppm to 10ppm enzyme,
Occurs in the wash solution at a product use concentration of approximately 0.17%.

Although the invention has been described in terms of specific embodiments, it may be modified to suit the invention generally in accordance with the principles of the invention and as known or conventional in the art to which the invention pertains. Any modification, use or use of the invention which has been carried out and may be applied to the essential features described above and which includes departures from the disclosure which is within the limits of the scope of the invention and the claims. It is intended as a diagram to cover all applications.

[Brief explanation of the drawing]

FIG. 1 is a map of the 4.3 kb EcoRI fragment of plasmid (7), designated pSN E'4. In Figure 1, the region shaded with diagonal lines represents the signal peptide codon (codons -22 to +1), and the region marked with dots represents the coding region of the mature polypeptide designated lipase 1 (codons +1 to +258). The ATG open codon and TAA stop codon are also shown. Figure 2 shows Pseudomonas (PscudoIIlonas).
) Lipase 1 gene of E. coi
i) Map showing expression vectors. The dotted region represents the coding region of the lipase signal sequence with 22 amino acids. The shaded region represents the coding region of the mature lipase protein. Transcription begins at the ATG start codon and proceeds in the direction indicated by the arrow to the TAA stop codon. The dark areas on either side are 5′
- and 3'-represent untranslated regions. Applicant's agent: Sato-OFIG, l.

Claims (1)

[Scope of Claims] 1. An enzymatic perhydrolysis system that generates peracid on the spot, which (a) has hydrolase activity and is composed of Pseudomonas putida ATCC5;
(b) a substrate hydrolysable by the enzyme of (a); (c) reacting with (a) and (b) to make the substrate soluble; a peroxygen source that produces peracid in the presence of an aqueous solution of 2. A patent in which the substrate has a structure ▲A mathematical formula, a chemical formula, a table, etc.▼ (However, R is a substituent having at least one carbon atom, and X is a functional residue or a hydrocarbon group) The enzymatic perhydrolysis system according to claim 1. 3. The enzymatic perhydrolysis system according to claim 2, wherein X consists of a functionalized polyol or polyether. 4. Enzymatic perhydrolysis system according to claim 2, wherein X has at least one carbon atom and at least one functional group. 5. The substrate of (b) has (i) structure ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (However, R_1 is C_1 to C_1_2, R_2 is C_1 to C_1_2 or H, and R_3 is C_1 to
(ii) Ethylene glycol derivatives having the structure ▲ Numerical formula, chemical formula, table, etc. ▼ (where n is 1 to 10 and R_1 is as defined above); (iii) Claim 4 selected from the group consisting essentially of propylene glycol derivatives having the structure ▲ Numerical formula, chemical formula, table, etc. ▼ (However, R_1 and n are as defined above) The enzymatic perhydrolysis system described in Section. 6. The enzymatic perhydrolysis system according to claim 5, wherein the substrate is normally incapable of substantial chemical perhydrolysis. 7, R_1 is C_6 to C_1_0, R_2 is C_
6~C_1_0 or H, and R_3 is C_6~C_
1_0 or H, the enzymatic perhydrolysis system according to claim 5. 8. The enzymatic perhydrolysis system of claim 1, wherein the substrate is normally insoluble in an aqueous solution and the aqueous solution that solubilizes the substrate contains an emulsifier. 9. The emulsifier comprises a water-soluble polymer, a cationic, nonionic, anionic, amphoteric or zwitterionic surfactant, a bile acid, or a mixture of any of the foregoing. The enzymatic perhydrolysis system described. 10. The enzymatic perhydrolysis system according to claim 8, further comprising a buffer, said buffer having a carbonate, phosphate, silicate, borate or hydroxide. 11. The enzymatic perhydrolysis system according to claim 1, wherein the substrate comprises triglyceride. 12. The enzymatic perhydrolysis system according to claim 1, wherein the enzyme is cloned from a gene expressing this enzyme and has the following amino acid sequence. [There is an amino acid sequence] 13. The enzymatic perhydrolysis system according to claim 12, wherein the substrate is trioctanoin or tridecanoin. 14. The enzymatic perhydrolysis system according to claim 12, wherein the substrate has at least one glyceride residue. 15. The enzymatic perhydrolysis system of claim 14, wherein the glyceride substrate is selected from the group consisting essentially of diglycerides and triglycerides. 16. A method for bleaching a substance, the process comprising: contacting the substance with an aqueous solution, comprising: (a) Pseudomonas putida;
putida) ATCC 53552 and (b) Structure ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (However, R is a substituent having at least one carbon atom, and X is a functional residue or a functionalized ester having a hydrocarbon group) which is hydrolyzable by the enzyme of (a); and (c) (a) and (b).
A method comprising the steps of combining an aqueous solution with an enzymatic perhydrolysis system having a peroxygen source which reacts with a peroxygen source to generate a peracid in situ. 17. The method of claim 16, wherein the substrate of (b) is normally not capable of substantial chemical perhydrolysis. 18. The method of claim 16, wherein X comprises a functionalized polyol or polyester. 19. The method of claim 16, wherein X has at least one carbon atom and at least one functional group. 20. The substrate of (b) has (i) Structure ▲ Numerical formula, chemical formula, table, etc. ▼ (However, R_1 is C_1 to C_1_2, R_2 is C_1 to C_1_2 or H, and R_3 is C_1 to
(ii) Ethylene glycol derivatives having the structure ▲ Numerical formula, chemical formula, table, etc. ▼ (where n is 1 to 10 and R_1 is as defined above); (iii) Claim 19 selected from the group consisting essentially of propylene glycol derivatives having the structure ▲ Numerical formula, chemical formula, table, etc. ▼ (provided that R_1 and n are as defined above) The method described in section. 21, R_1 is C_6 to C_1_0, R_2 is C
_6~C_1_0 or H, and R_3 is C_6~C
21. The method according to claim 20, wherein _1_0 or H. 22. The method of claim 16, wherein the substrate is normally insoluble in an aqueous solution, the system further comprises an emulsifier, and the enzyme has an amino acid sequence as follows: [The amino acid sequence is] 23. The emulsifier consists essentially of a water-soluble polymer, a cationic, nonionic, anionic, amphoteric or zwitterionic surfactant, a bile acid, or a mixture of any of the above. 23. The method of claim 22, wherein the method is selected from the group consisting of: 17. The method of claim 16, further comprising a buffer selected from the group consisting essentially of: 24, carbonate, phosphate, silicate, borate or hydroxide. 25. The method of claim 17, wherein the substrate is selected from the group consisting essentially of diglycerides and triglycerides. 26. The method of claim 16, further comprising an emulsifier capable of at least maintaining or improving the peracid yield achieved by the system in the absence of an emulsifier.